1. Field of the Invention
This invention relates to the control of viral infections of fish, and more specifically to vaccines for such control.
2. General Discussion of the Background
Viral diseases of fish are increasing throughout the world, threatening the future of both ocean and freshwater fisheries. The problem of infection is particularly acute among fry in hatcheries where populations are dense and diseases are rapidly transmitted.
For example, in recent years, an alarming increase in the incidence of infectious hematopoietic necrosis (IHN) virus in fish has been observed at trout and salmon hatcheries. Since its initial isolation in 1953, IHN virus has spread throughout the Pacific Coast of Northern America. It is now found in 60- to 90-percent of spawning sockeye salmon in Alaska.
Efforts to control the spread of IHN virus in the Columbia River Basin alone resulted in the destruction of more than 14 million eggs and fish in 1980-82. The magnitude of the losses were such that fish production quotas could not be met at certain hatcheries. Thus, IHN disease is costly and threatens the continued existence of valuable fisheries.
IHN virus most severely affects fry and juvenile salmon or trout. The infected fish generally appear normal until shortly before death when they become darker and show hemorrhages at the bases of fins and at the throat. The kidneys and spleen are the most severely affected organs.
An epizootic of IHN is characterized by an abrupt and high mortality among the infected population. In fish up to two months of age, the mortality can exceed 90 percent. Fish from two to six months of age have a mortality rate of 50 percent and in 6- to 12-month-old fish, the mortality rate is 10 percent. The disease has not been seen in fish two years of age or older.
The actual route of IHN virus transmission in nature is unknown. However, experimental studies have shown that the virus can be transmitted through water from infected fish as well as by ingestion of diseased fish carcasses. Congenital transmission of the virus from adult carrier fish to their young is thought to be the principal mode of transmission. The virus is frequently found in the ovarian or seminal fluid of carrier fish at the time of spawning. Thus, IHN virus is believed to be transmitted on eggs as an external contaminant.
Presently, the only practical method available to control the spread of IHN virus is the removal and destruction of all infected fish, the disinfection of all ponds and equipment, and the restocking of the hatchery with virus-free eggs. Since IHN virus is believed to be transmitted with eggs as an external contaminant, eggs are disinfected with iodophore treatment for 10 minutes at pH 6.0. The effectiveness of this treatment is still controversial since eggs are water-hardened before the iodophore bath. The water hardening process may allow virus to enter the egg and thus make the virus insensitive to iodophore treatment.
Another method of disease control is the rearing of susceptible fish at water temperatures above 15.degree. C. This method is not usually feasible on a large scale since 15.degree. C. water temperatures are difficult to easily and economically obtain.
Killed virus vaccines and live, attenuated virus vaccines have been used as prophylactic measures in man for years. However, both types of vaccine give rise to undesirable side effects. In both preparations, there are nonviral proteins, e.g. cellular material, that may induce undesirable auto-allergic antibodies.
Furthermore, extensive work must be done to insure the safety of live, attenuated viral vaccines. Before such vaccines may be released for widespread use, 1) the degree and stability of attenuation must be established by extensive laboratory and clinical trials; 2) the attenuated virus strain should exhibit low communicability under field use; 3) reliable "marker" tests must be developed to allow differentiation of virulent and attenuated virus strains; 4) the vaccine preparation must be free from other viral contaminants; 5) the attenuated virus strain must also be nononcogenic; and 6) the attenuated virus must not establish persistent infections with consequent chronic disease in the host. These requirements usually make the mass production of a live, attenuated virus vaccine too costly for animal species other than humans.
Despite the low likelihood of developing a practical vaccine, both killed and live modified types of IHNV (IHN virus) vaccines have been tested experimentally (Rohevec, et al., Natl. Sci. Council Symp., Series No. 3, Natl. Sci. Council, Taipei, Taiwan, pp. 115-121, 1981), because none of the other available treatments is very successful.
An attenuated strain of IHN virus was developed in 1974 (McMichael, Ph.D. Diss., Oregon State University, 1974). The vaccine was produced by transferring an Oregon sockeye salmon isolate on steelhead trout cells in tissue culture for more than forty passages. The attenuated virus immunizes sockeye salmon fry or juveniles after contact with the virus for 48 hours. In most trials, 90 percent or more of the vaccinated fish were protected against a fatal infection with the wild-type virus for as long as 110 days (Tebbit, Ph.D. Diss., Oregon State University, 1976; Fryer, et al., CRC Crit. Rev. Micro., 7:287-344, 1976). Attenuated strains are not, however, in commercial use since they are virulent in rainbow trout and the reversion frequency from nonpathogenic to pathogenic wild-type virus has not been determined.
Amend and Smith, J. Fish. Res. Bd. Can., 31:1371-1378, 1974, showed that rainbow trout will develop neutralizing antibody within 54 days after injection with killed virus. Sera obtained from these fish were protective when juvenile fish were passively immunized and experimentally challenged with virulent virus.
Although the experimental tests of Amend and Smith have shown that a killed IHN viral vaccine is effective, the cost and labor-intensive procedures required for preparing a killed viral vaccine make it impractical for widespread use at fish hatcheries.
Thus, no practical vaccine, antiviral chemical, or passive immunization therapy has heretofore been available to eliminate or ameliorate a viral disease in fish.
A recently developed method for producing viral vaccines is the biosynthesis of a viral subunit component. In this method, the gene for the viral protein is isolated and provided with new regulatory signals appropriate for its expression in a new host organism. For example, the gene for the major surface antigen of hepatitis B virus has been directly expressed both in the yeast Saccharomyces cerevisiae (Valenzuela, et al., Nature, 298:347-350, 1982) and in simian cells (Liu and Levinson, in Eucaryotic Viral Vectors (V. Gluzman, ed.), Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1982). Similarly, vaccines have been developed to resist human diseases caused by herpes 1 and 2 viruses and polio-virus.
Kleid and coworkers have reported the synthesis in Escherichia coli of an immunogenic fusion protein between an antigen, VP.sub.3, of foot-and-mouth disease virus and a protein derived from the E. coli pathway for tryptophan biosynthesis (Kleid, et al., Science, 214:1125-1129, 1981).
One possible method for producing a virus-subunit vaccine is to use a known nucleotide sequence to deduce the amino acid sequence of an immunogenic viral protein. Then, short polypeptides are selected from the deduced sequence and chemically synthesized, attached to carrier proteins of known immunogenicity, and injected in an immunopotentiating adjuvant. As illustrated for the cases of hepatitis virus and foot-and-mouth disease virus (Lerner, et al., Proc. Natl. Acad. Sci. USA, 78:3403-3407, 1981; Bittle, et al., Nature, 298:30-33, 1982; Sutcliffe, et al., Science, 219:660-666, 1983), in some instances, a given linear array of amino acids can be an effective antigenic domain; the native three-dimensional structure of the intact protein is not required to evoke a spectrum of reactive and neutralizing antibodies in vivo.
One area of recent activity on viral subunits has been with the rabies virus which, like IHNV, is also a bullet-shaped, enveloped rhabdovirus (Hill, et al., J. Gen. Virol., 27:369-378, 1975). Although both rabies and IHN viruses produce G proteins, the viruses do not immunologically cross-react (Hill, et al., supra), indicating that G proteins from the two viruses are appreciably different.
The full-length coding sequence of one strain of rabies G mRNA has been determined and its sequence or portions thereof expressed in a bacterial host (Yelverton, et al., Science, 219:614-620, 1983; Goeddel and Yelverton, European Patent No. 0117657, 1984). However, no protection against rabies infection using the bacterially synthesized material has yet been reported (Kieny, et al., Nature, 312:163-166, 1984). Kieny, et al. (supra), constructed a recombinant vaccinia virus (VV) whose genome contained the rabies glycoprotein cDNA. Inoculation of rabbits and mice with live recombinant VV induced production of anti-rabies antibodies and protection against subsequent challenge with live rabies virus. These results suggest that some required rabies G-protein post-translational processing occurs in eucaryotic cells, but not bacterial cells. For rabies virus, this is a serious limitation on large-scale vaccine production because culturing sufficient numbers of eucaryotic cells on their required solid supports is difficult and labor-intensive. Bacterial culture in suspension, on the other hand, would be much simpler to perform on a virtually unlimited large scale.
IHNV is different from other rhabdoviruses in other respects. First, essentially no homology exists between mRNAs from IHNV and viral hemorrhagic septicemia (VHS) virus, another salmonid rhabdovirus (McAllister and Wagner, J. Virol., 22:839-843, 1977). Second, the IHNV viral genome is unique in encoding six, rather than the five, viral proteins normally associated with rhabdoviruses (Kurath and Leong, J. Virol., 53:462-468, 1985). Hence, an effective process for producing an anti-IHNV vaccine comprised of immunogenic IHNV G protein or portions thereof, suitable for administering to fish on a large scale at low cost, and that contains no live or attenuated viruses of any sort, cannot be extrapolated from work performed with other viruses, even those within the same taxonomic group.
Thus, despite advances with other viruses, until the present invention, there has been no successful attempt to create a subunit vaccine suitable for practical administration to large numbers of fish.